Abstract
2-hydroxyglutarate (2HG) is a potent competitor of α-ketoglutarate (α-KG) and can inhibit multiple α-KG dependent dioxygenases that function on the epigenetic modifications. The accumulation of 2HG contributes to elevated risk of malignant tumors. 2HG carries an asymmetric carbon atom in its carbon backbone and differentiation between D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) is crucially important for accurate diagnosis of 2HG related diseases. Here we developed a strategy by chiral derivatization combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive determination of D-2HG and L-2HG enantiomers. N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was used to derivatize 2HG. The formed diastereomers by TSPC labeling can efficiently improve the chromatographic separation of D-2HG and L-2HG. And derivatization by TSPC could also markedly increase the detection sensitivities by 291 and 346 folds for D-2HG and L-2HG, respectively. Using the developed method, we measured the contents of D-2HG and L-2HG in clear cell renal cell carcinoma (ccRCC) tissues. We observed 12.9 and 29.8 folds increase of D-2HG and L-2HG, respectively, in human ccRCC tissues compared to adjacent normal tissues. The developed chiral derivatization combined with LC-ESI-MS/MS analysis offers sensitive determination of D-2HG and L-2HG enantiomers, which benefits the precise diagnosis of 2HG related metabolic diseases.
Highlights
One of the examples of the role of metabolism in cancer is the recently identified onco-metabolite with putative oncogenic property, 2-hydroxyglutarate (2HG), which is typically generated by mutated isocitrate dehydrogenase 1 and 2 (IDH1/2)
To achieve good chromatographic separation of the enantiomers of D-2HG and L-2HG, here we employed TSPC that harbors a chiral carbon to derivatize D-2HG and L-2HG to form diastereomers (Fig. 2A), which may have slightly different interaction with stationary phase due to their different steric configuration and offer the possibility for these diastereomers to be separated by LC
The carboxyl group in 2HG will not react with TSPC under this reaction conditions and we did not detect any products formed through the reaction between the carboxyl group of 2HG and TSPC, which is due to the carboxyl group is a weaker nucleophile than hydroxyl
Summary
One of the examples of the role of metabolism in cancer is the recently identified onco-metabolite with putative oncogenic property, 2-hydroxyglutarate (2HG), which is typically generated by mutated isocitrate dehydrogenase 1 and 2 (IDH1/2). We developed a novel strategy by chiral derivatization combined with liquid chromatography/tandem mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive determination of onco-metabolites D-2HG and L-2HG enantiomers.
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